2 4 6 tripyridyl s triazine tptz

FeSO₄ × 7H₂O was used for a calibration curve. The compound was dissolved in methanol and diluted with water to obtain concentrations of 0.0125, 0.025, 0.05, 0.10, 0.15, 0.20, and 0.30 mg/mL. From the phenol samples, 1 mg/mL methanolic solutions were prepared and diluted with water to finally obtain seven concentrations: 0.1, 0.08, 0.06, 0.04, 0.03, 0.02, 0.01 mg/mL, respectively.
The assay was performed according to Benzie and Strain [29 (link)], with some modifications. The FRAP working solution was prepared prior to the start of the analysis: 0.3 mol acetate buffer (pH 3.6), 0.01 mol TPTZ (2,4,6-tripyridyl-s-triazine; Sigma-Aldrich, Poznań, Poland) in 0.04 mol HCl (POCH, Lublin, Poland) and 0.02 M FeCl₃ × 6H₂O in water (iron (III) chloride hexahydrate; Chempur, Poland) were mixed in a volumetric ratio of 10:1:1 and protected from light. Next, 75 µL of the phenol solutions or FeSO₄ × 7H₂O solutions were mixed with 2.25 mL of the FRAP working solution and 225 µL of water. The obtained mixtures were incubated at 37 °C for 30 min, and their absorbance was measured at 593 nm. Deionized water with a FRAP solution was used as a blank.

Platinum (IV) chloride and palladium (II) acetate were obtained from BLD Pharmatech Co., Limited, Cincinnati, OH, USA. TPTZ (2,4,6-tripyridyl-s-triazine) and FeCl₃.6H₂O were purchased from Sigma Aldrich, St. Louis, MO, USA. Streptomycin, penicillin, fetal bovine serum, trichloroacetic acid (TCA), Dulbecco’s Modified Eagle’s Medium (DMEM) for sulforhodamine B (SRB) assay, and Tris(hydroxymethyl)aminomethane were purchased from Lonza, (Basel, Switzerland).

FRAP assay was performed following the protocol of Abdelaleem and Elbassiony (2021) [21 (link)] with minor adaptations. In particular, ascorbic acid was used as antioxidant standard and prepared in seven dilutions from 1000 µL to 0 µL. a) Acetate buffer (300 mM; pH 3.6) was prepared using 2.69 g sodium acetate trihydrate (Sigma Chemical Co. (St. Louis, MO, USA)) in 16 mL of glacial acetic acid and made the volume to 1.0 L with distilled water. b) TPTZ (2, 4, 6-tripyridyl-s- triazine), provided by Sigma Chemical Co. (St. Louis, MO, USA), was obtained dissolving 31.2 mg in 10 mL of 40 mM of HCl. c) FeCl₃ (Sigma Chemical Co. (St. Louis, MO, USA)) was obtained dissolving 0.054 g in 10 mL of distilled water. Then, 10 µL of each sample was added to 300 µL of FRAP reagent and incubated at RT for 10 min in the dark and read at 595 nm. Appropriate solvent blanks were run in each assay. Values were expressed in terms of acid ascorbic equivalent (mg AAE/100 g).

Sephadex LH-20 was purchased from Amersham Bioscience. All solvents used in the experiments were purchased from LAB-SCAN. Chlorhydric acid (HCl), trifluoroacetic acid (TFA), butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulphonic acid)-1,2,4-triazine (ferrozine), iron (II) chloride tetrahydrate (FeCl₂·4H₂O), iron (II) chloride (FeCl₂), iron (III) chloride (FeCl₃), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and TPTZ (2,4,6 tripyridyl-s-triazine) were purchased from Sigma-Aldrich (Steinheim, Germany).

The ferric reducing antioxidant power assay is similar to the procedure described by Benzie and Strain [75 (link)]. This assay is based on the reduction of ferric-tripyridyltriazine complex to its ferrous in the presence of antioxidants. The following reagents were used: 2.5 mL of a 10 mmol/L TPTZ (2,4,6- tripyridyl-s-triazine, Sigma-Aldrich, Johannesburg, South Africa) solution in 40 mmol/L HCl plus 2.5 mL of 20 mmol/L FeCl₃ and 25 mL of 0.3 mol/L acetate buffer and maintained at pH 3.6 was prepared freshly and warmed at 37 °C. Aliquots of 40 μL of the sample supernatant were mixed with 0.2 mL distilled water and 1.8 mL FRAP reagent. After incubation at 37 °C for 10 min, we employed spectrophotometric method to determine the absorbance of the reaction mixture at 593 nm. The standard solution was 1 mmol/L of FeSO₄, and the final result was expressed as the concentration of antioxidants having a ferric reducing ability equivalent to 1 mmol/L FeSO₄.

Ethidium bromide, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Ferric chloride, Nicotinamide adenine dinucleotide (NADH), Phenazine methosulphate, Ascorbic Acid, 2-Thiobarbituric Acid and L-Ascorbic acid were obtained from HiMedia Pvt. Limited. Mumbai. 2-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS), TPTZ (2,4,6-tripyridyl-s-triazine), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Deoxyribose were purchased from Sigma Chemical Co. (St Louis, MO, USA). pBR322 plasmid DNA was purchased from Genei Pvt. Ltd., Banglore (India). All other chemicals used in the present experimental study were of AR grade.

Sephadex powdered matrix (G-150; GE Healthcare UK Limited) with bead-diameter - 20-300 μm; fractionation range - 5 to 300 kDa, TPTZ 2, 4, 6-tripyridyl-s-triazine and ferric chloride (hexahydrate) from Sigma, USA; protein molecular mass standard (Bangalore Genei (Bangalore, India); sodium dodecyl sulphate (SDS), acrylamide and bisacrylamide (electrophoresis grade; Merck, Germany). Phosphate buffer saline(PBS)), The other chemicals and their sources of procurement were as follows: Dulbeco's Modified Eagle Medium (DMEM), trypsin phosphate versene glucose (TPVG) solution Trypsin and methylthiazolyldiphenyltetrazolium bromide (MTT) (Hi Media Laboratories Pvt. Ltd. Mumbai, India), Fetal bovine serum (FBS; Biosera, East Sussex, UK) and dimethyl sulfoxide (DMSO; Sisco Research Laboratories Pvt. Ltd. Mumbai, India).